Organ transplantation is the established treatment for end-stage organ disease. However, there exists a worldwide shortage of organs available for transplantation. In the USA alone, there are currently approximately 60,000 people waiting for organ transplants and 4000 die every year before a transplant can be performed [1] with the waiting list artificially constrained in size due to the limited type and number of donor organs available. Many additional patients that could benefit from organ transplantation are not listed. Xenotransplantation, the use of animal organs and cells for transplantation into humans, has the potential to alleviate this shortage [2]. Although some consideration has been given to the use of non-human primates as donors, swine are now accepted as the animal of choice for xenotransplantation due to a number of practical, financial, and ethical reasons [2].
The present invention solves problems of xenotransplantation by providing a herd of swine, preferably miniature swine, and preferably inbred at the MHC (Major Histocompatibility Complex) locus, for xenotransplantation. In accordance with the invention, miniature swine were chosen for use herein because they exhibit several attractive characteristics. Like their domestic counterparts, miniature swine reach sexual maturity at an age of 4-5 months and give birth to multiple offspring (3-10 per litter). In addition they have an estrous cycle every 3 weeks, permitting breeding throughout the year. Miniature swine reach an adult size of 200-300 pounds (a size comparable to adult humans) in contrast to domestic swine that attain weights of over 1000 pounds. This difference is important in the programmed growth of a transplanted porcine organ. Furthermore, the animals developed herein have been housed under defined conditions and have an extensive medical history associated with them.
Although xenotransplantation clearly has the potential to alleviate much suffering, certain safety concerns are associated with the procedure [3,4]. These concerns can be divided into infectious risk to the patient, and particularly with xenotransplantation the risk to contacts of the recipients. The most serious concern is the possibility of transmission of microorganisms from the xenograft to the recipient and the subsequent emergence of a new human infection and possibly disease. A major reason that pigs are considered the donor animal of choice in preference to non-human primates is due to the reduced microbiological burden that they carry. Risk of transmission of microorganisms is not unique to xenotransplantation. Many cases have been documented of transmission of organisms causing disease during allotransplantation procedures [5].
Cross-species infection (zoonosis), in comparison to transmissions confined within a species, is of particular concern because the behavior of an organism once it crosses the species barrier cannot be predicted by its pathogenicity in its natural host. Organisms benign in their natural host can cause significant morbidity in a zoonotic scenario. This is exemplified by the potentially fatal infections of humans with the Nipah virus of pigs, herpes B virus of primates and hantavirus of rodents [6-8]. Secondly, the consequences of transmission of an organism might not stop solely with the xenograft recipient. The possibility exists that once contracted by the xenograft recipient, the organism may be transmissible to contacts of the recipient. Thus the control and monitoring of recurrent infection during xenotransplantation procedures is viewed as an important public health issue [2]. In addition, although it may be possible to detect an infection of the recipient by a microorganism quickly and before manifestations of disease become apparent, effective treatments may not have been developed because in almost all cases no previous infections by this organism will have been documented. As a consequence, the prognosis of infection by zoonotic microorganisms transmissible through xenotransplantation are essentially unknown and thus the organisms specified for removal include all organisms known to produce disease in either man or pig.
The microorganisms that could be transferred along with the organ vary in their potential to establish an infection in the recipient and therefore their importance to xenotransplantation. Raising animals under germfree methodologies such as described in Ratcliffe and Fodham (1987) Laboratory Animals 21: 53-59 facilitates removal and prevention of the reoccurrence in the herd of many of the organisms considered a potential risk to xenotransplantation. The greatest risk of infection is probably with those organisms that have an ability to be transferred as an asymptomatic latent entity within the organ. These problematic characteristics have made endogenous retroviruses (ERV) and herpesviruses the focus of attention by regulatory agencies and the production of animals clear of such organisms a priority. In accordance with the present invention, such problems are wholly, or, at least, partially, solved by the production of certain animals within a herd (such as the miniature swine used herein) with a unique advantage over other breeds of pig with respect to endogenous microorganisms.
Endogenous retroviruses (ERVs) have been identified as a constituent of the normal DNA of every vertebrate species tested including pigs and humans. A unique attribute of the normal retrovirus lifecycle is the stable integration of genetic material into the host cell chromosomal DNA. Where the host cell is a germ-line cell, the viral nucleic acid material (or provirus) will subsequently be inherited by all offspring in a manner typical of any other Mendelian gene. Consequently, if the presence of a particular provirus in the DNA of a germ-line cell places the offspring at a selective disadvantage it would not be expected to survive over evolutionary time periods and this ERV is not expected in the ongoing gene pool. Thus, the ERV present in the germ-line of animals today tend not to be pathogenic for their own species. Individual ERV loci also tend to be replication defective due to mutations present in their genome. However, the potential exists for individual defective loci to interact by complementation and recombination to form infectious virus. The lack of pathogenicity of ERV for their normal host species leaves no room for complacency because the very same viruses can change their pathogenicity when interspecies transmission occurs. For example, Gibbon Ape Leukemia Virus is thought to have evolved following infection of gibbons with a nonpathogenic endogenous virus of mice [9]. It now spreads in captive gibbons causing lymphoid and myeloid malignancies. Furthermore, tumorogenic properties due to the activities of ERV have been observed in non-human primates undergoing retroviral gene therapy treatments [10].
Pig endogenous retroviruses (PERV) represent a unique and possibly the most important safety concern for xenotransplantation. Unlike other infectious organisms that a pig may carry, these viruses are not transmitted between animals as an infectious agent but rather are inherited by all animals as part of their germ-line DNA. The viruses are present in all breeds of swine and form part of the normal DNA present in every cell. Approximately 50 copies of the virus are present in every cell, and as such cannot be completely removed by conventional breeding techniques. As a consequence, these DNA sequences are certain to be present in all swine cells used for xenotransplants.
The production of PERV and transmission to human cells has heretofore been studied in vitro in great detail and it has been shown that two families of PERV (PERV-A and PERV-B) can replicate in human and porcine cells [11,12]. A third family (PERV-C) can replicate in porcine cells only [11,12]. The human kidney cell line 293 is clearly the most permissive for PERV replication and has thus been selected for co-culture assays [11]. Retroviruses use cell surface molecules to act as receptors and mediate virus entry. The expression pattern of these cellular receptors indicates that the three families of PERV use independent receptors, not used by other infectious retroviruses [12]. Significantly, some cells that are permissive for virus entry do not support virus replication [11, 12]. PERV production has been examined from pig cell lines and also from primary cell cultures from several pig breeds [13]. All swine cells, with the single exception of the cell line ST-IOWA, appear to produce PERV capable of infecting and replicating in human cells. Breeds of pig tested to date include the NIH minipig, Yucatan, multiple land breeds, and the animals currently being used in clinical trials. In short, the use of a specific-pathogen-free (SPF) breeding program would eliminate most pathogens that might be transmitted during xenotransplantation. However, pathogens such as ERV that are transmitted through the germ-line would not be eliminated and the recent evidence cited above demonstrates that certain PERVs are capable of infecting human cells so that one of the potential risks from the use of pig organs is the transmission of such pathogens.
Studies into PERV are complicated by the predominance of defective copies of the virus, which are very closely related to replication competent copies of the virus. In order for a single locus to encode for replication competent virus, a genomic copy of PERV must contain functional LTR's and open reading frames for gag, pol and env and it is therefore necessary to be able to identify these loci specifically. In addition, RNA from defective loci may once pseudotyped into human cells, recombine with other PERV loci to form replication competent retrovirus (RCR). Sequencing of isolated single genes, although it may identify open reading frames, cannot be extrapolated as inferring competence of a locus for replication.
The regulatory boards governing xenotransplantation trials in both the USA and UK at one point halted clinical trials in reaction to the initial discovery that human cells could be infected by PERV. Since then a number of published reports have presented data indicating that a limited number of patients transiently exposed to relatively small numbers of porcine cells and organs show no evidence of PERV transmission [14-16]. Limited clinical trials have recommenced with the requirement of chronic screening of recipients for PERV infection. The possible lifelong screening currently required clearly places not only a significant monetary cost on the xenotransplantation procedure, but also a practical burden on the patient due to the repeated monitoring procedures. Consequently, use of animals devoid of transmissible PERV as xenograft donors is not only safer but also affords reduced costs of post-transplant screening procedures.
In accordance with the present invention, the inbred herd of swine used herein contains a novel subgroup of animals that does not produce PERV capable of replication in human cells even by recombinantion. Based on the disclosure herein, specific breeding can be performed to eliminate all genetic components that can contribute to form a human-tropic replication competent PERV present in the pigs.